Method for detecting a progredient chronic dementia, and associated peptides and detection reagents

ABSTRACT

The invention relates to defined peptides and the quantitative determination thereof in body fluids of patients suffering from progredient chronic dementia, in relation to the concentration of said peptides in a control group. The inventive peptides come from a protein precursor having the corresponding gene, are processed in a specific manner, and are optionally post-translationally modified, especially phosphorylised. An increase in the concentrations of these peptides or the corresponding non-processed protein indicates progredient chronic dementia. Progredient chronic dementia is detected by identifying the peptides and/or the protein individually or in combinations. The invention also relates to the use of said peptides for controlling the course of progredient chronic dementia and for the prognosis of progredient chronic dementia, especially for complementing or replacing mini-mental scores, and for developing therapeutic agents to combat progredient chronic dementia such as Alzheimer&#39;s disease.

[0001] The invention relates to a method for detecting progressive,chronic dementia diseases or a predisposition to such diseases, inparticular an alternative or supplementary method to the determinationof the mini-mental score by determining the severity of the dementia.For this purpose, the concentration of particular peptides and bodyfluids or other samples from the patient is determined. The inventionfurther relates to peptides which have been found for determining thepresence and/or the degree of the progressive, chronic dementia disease.

[0002] The invention additionally relates to detection reagents such asantibodies and nucleic acids and the like, via which these peptides orthe corresponding nucleic acids can be detected. The invention furtherrelates to pharmaceutical applications which comprise OPN, OPN peptides,OPN antibodies, OPN nucleic acids, OPN protein antagonists, or OPNprotein agonists, OPN peptide agonists or OPN peptide antagonists, forthe therapy or prophylaxis of neurological diseases, especially ofAlzheimer's disease. The invention further relates to methods foridentifying patients with neurological diseases, especially Alzheimer'sdisease, who are suitable for taking part in clinical studies toinvestigate these diseases.

[0003] Dementia diseases represent an increasing problem inindustrialized countries because of the higher average life expectancy.Dementia diseases are in most cases incurable and make long-term care ofthe patients necessary. About half of these patients receive inpatientcare. More than 60 dementia diseases are known, including diseasesassociated with manifestations of dementia.

[0004] However, Alzheimer's disease (AD) accounts for about 65% ofthese, and the diagnosis and therapy thereof is therefore of greatimportance. Besides Alzheimer's disease, the following non-Alzheimer'sdementias are known, inter alia: vascular dementia, Lewy body dementia,Binswanger dementia, and dementia diseases which occur as concomitanteffect of other disorders such as Parkinson's disease, Huntington'sdisease, Pick's disease, Gerstmann-Straussler-Scheinger disease,Kreuzfeldt-Jakob disease etc.

[0005] Alzheimer's disease is a neurodegenerative disease distinguishedby the following symptoms: decline in intellectual abilities, confusionand diminished ability to look after themselves. A greatly restrictedshort-term memory in particular is characteristic of Alzheimer'sdisease, whereas the patient's memories of the distant past, e.g. ofhis/her own childhood, is impaired far less by the disease. There aremorphological changes in the brain manifested inter alia in the form ofamyloid deposits and degenerated nerve cells. The morphological changescan be diagnosed histologically after the patient's death and are as yetthe only reliable detection of the disease. These histopathologicaldiagnoses are based on criteria fixed by the Consortium to Establish aRegistry for Alzheimer's Disease (CERAD). The following criteria-baseddiagnostic systems are currently used to diagnose Alzheimer's disease:the International Classification of Diseases, 10th revision (ICD-10),the Diagnostic and Statistical Manual of Mental Disorders, 4th edition(DSM-IV) of the American Psychiatric Association, and the Work Groupcrieria drawn up by the National Institute of Neurological andCommunicative Disorders Association NINCDS-ADRDA.

[0006] These systems use a number of neuropsychological tests in orderto diagnose Alzheimer's disease, but not objectively measurable clinicalparameters. It is of particular interest to establish the current degreeof severity of the disease, which can take place for example bydetermining the mini-mental score. The mini-mental score is determinedwith the aid of a mini-mental state examination (MMSE), a psychologicaltest. This makes it possible inter alia to observe the course of thedisease and the efficacy of any therapies. However, Clark et al wereable to show that, for example, determination of the mini-mental scorehas only limited validity for determining the course of Alzheimer'sdisease because large measurement inaccuracies and wide variations inthe level of the score occur [1]. The provision of a reliable,clinically measurable parameter which can supplement or replace themini-mental score for determination of the course of progressive,chronic dementia diseases such as, for example, Alzheimer's disease istherefore of great medical, and thus also economic, importance.

[0007] At present, no causal therapy is available for the treatment ofAlzheimer's disease. The disease is merely treated symptomatically, e.g.by administration of neurotransmitters such as acetylcholine. Furtherpossible therapeutic strategies being tested at present are theadministration of antioxidants, of radical scavengers, of calciumchannel blockers, of anti-inflammatory substances, of secretaseinhibitors, of anti-amyloid antibodies etc., and immunization againstamyloid peptides. However, no causal therapy of this disease is yetpossible.

[0008] The invention is based on the object of avoiding the prior artdisadvantages in the diagnosis of Alzheimer's disease and of providing amethod which can be used early and reliably for detecting chronicdementia diseases, especially Alzheimer's disease.

[0009] Novel therapies for the treatment of Alzheimer's disease are madepossible for the first time by this diagnosis.

[0010] Definitions:

[0011] OPN Proteins or Peptides Corresponding to Accession No. X13694:

[0012] The peptide derived from the nucleic acid sequence X13694 is alsoreferred to as OPN protein and includes all naturally occurring alleles,mutants and polymorphisms of OPN proteins, and tissue-specificallyexpressed OPN variants. Included in particular are also the OPN variantswhich occur because of diseases or as a result of neurological diseases,especially chronic dementia diseases, especially Alzheimer's disease.There is inclusion both of OPN proteins with and without signalsequence, proforms of OPN proteins which have not yet been processed,and already processed OPN proteins, soluble OPN proteins andmembrane-associated OPN proteins, where the membrane-associated OPNproteins may be linked both via transmembrane amino acid sequences to acell membrane or organelle membrane and via a post-translationalmodification, e.g. a glycosyl-phosphatidyl-inositol (GPI) anchor. Alsoincluded are variations of the OPN sequence which [lacuna] byalternative splicing, by alternative translation starting andtermination points, by RNA editing, by alternative post-translationalmodifications, and other OPN protein variants arising through naturallyoccurring mechanisms.

[0013] DROPN Peptides:

[0014] OPN peptides and OPN peptide variants are referred to hereinafteras DROPN (“dementia related osteopontin”) peptides. DROPN peptides arederived from the OPN sequence X13694 mentioned at the outset.Alternatively, DROPN peptides may also be derived from other Gene Bankentries for osteopontin, such as, for example, AF052124, J04765, M83248,NM_(—)000583, U20758 or further OPN entries which will possibly also beadded in future. It is moreover possible for the OPN protein sequencespossibly to differ from the sequence of the Gene Bank entry with thenumber X13694, as is currently the case already for the Gene Bankentries AF052124, J04765 and NM_(—)000582. OPN sequence entries may alsobe present in other sequence databases different from “Gene Bank”.Consequently, DROPN peptides and OPN proteins need not agree exactlywith the sequence of the OPN protein corresponding to the entry in the“Gene Bank” sequence database with the accession No. X13694. Inaddition, DROPN peptides may include two point-mutated, two deleted ortwo additionally internally inserted amino acids, and N-terminal and/orC-terminal extensions. However, in these cases they must retain at least8 amino acids from the OPN protein sequence. The only amino acidssuitable as N— or C-terminal extensions are those occurring in the OPNprotein sequence at this sequence position in the OPN protein. Peptidesderived from naturally occurring OPN polymorphisms and from naturallyoccurring OPN mutants are also referred to as DROPN peptides as long asthey show at least 70% agreement with the OPN protein sequence (X13694).DROPN peptides may also exist with post-translational modifications suchas, for example, phosphorylations or N-terminal pryroglutamic acidresidues and/or in chemically modified form, preferably as peptideoxides. For example, DROPN-10 has been identified both asnon-phosphorylated and as phosphorylated peptide. DROPN-10 occurs, forexample, without phosphate group and with one, two, three, four or fivephosphate groups.

[0015] Chemically or Post-Translationally Modified Peptides:

[0016] A chemically or post-translationally modified peptide may consistboth of D- and of L-amino acids, and of combinations of D- and L-aminoacids and may occur naturally, be prepared recombinantly or synthesizedchemically. These peptides may additionally comprise unusual aminoacids, i.e. amino acids which do not belong to the 20 standard aminoacids. Examples of unusual amino acids are, inter alia:alpha-aminobutyric acid, beta-aminobutyric acid, beta-alanine,beta-aminoisobutyric acid, norvaline, homoserine, norleucine,gamma-aminobutyric acid, thioproline, 4-hydroxyproline,alpha-aminoadipic acid, diaminobutyric acid, 4-aminobenzoic acid,homocysteine, alpha-aminopenicillanic acid, histamine, ornithine,glycineproline dipeptide, hydroxylysine, proline-hydorxyprolinedipeptide, cystathionine, ethionine, seleno-cysteine. Possiblepost-translational or chemical modifications are, inter alia,modifications of amino acid sequences by the following structures:linkage of free cysteine to a cysteine in the peptide sequence, methyl,acetyl, farnesyl, biotinyl, stearoyl, palmityl, lipoyl, C-mannosyl,phosphorus and sulfate groups, glycosilations, amidations, deamidations,pyroglutamic acid, citrulline etc.

[0017] Nucleic Acids:

[0018] Nucleic acids are regarded as being DNA, RNA and DNA-RNA hybridmolecules both of natural origin and prepared synthetically or byrecombination. Also included are chemically modified nucleic acids whichcomprise modified nucleotides having high in vivo stability, such as,for example, phosphorothioates. Such stabilized nucleic acids arealready used in the application of ribozyme, antisense and triplexnucleic acid techniques.

[0019] Significance:

[0020] The term significant is used in the sense in which the termsignificance is used in statistics. In this patent application, an errorprobability of less than 90%, preferably 95% further preferably 99% isdefined as significant.

[0021] Sensitivity:

[0022] Sensitivity is defined as the proportion of patients with thedisease who acquire a positive diagnostic result in a diagnosis for thedisease, i.e. the diagnosis correctly indicates the disease.

[0023] Specificity:

[0024] The specificity is defined as the proportion of healthy patientswho acquire a negative diagnostic result in a diagnosis for the disease,i.e. the diagnosis correctly indicates that no disease is present.

[0025] It has surprisingly been found that in samples of body fluidsfrom patients suffering from Alzheimer's disease, especially in thecerebrospinal fluid, the concentration of certain peptides is changedgreatly relative to their concentration in control samples, and thusmakes detection of Alzheimer's disease possible. Changes in theconcentration of these peptides relative to their concentration incontrol groups indicate the presence of Alzheimer's disease and aretherefore suitable for detecting this disease with high sensitivity andspecificity. Modulation of the OPN protein or DROPN peptideconcentration with the aim of adjusting the patient to normal OPN orDROPN peptide concentrations can thus be used therapeutically.

[0026] To achieve the object, the invention includes a method fordetecting a neurological, in particular of a chronic dementia disease,in particular of Alzheimer's disease, or of a predisposition to such adisease by identifying one or more DROPN peptides or OPN peptides whichare derived from the sequence having the Gene Bank accession No. X13694,in a biological sample from an individual. Since these DROPN peptides orOPN peptides are presumably causally connected with the disease, thepresent invention also includes the use of these peptides for thetherapy of Alzheimer's disease or related neurological diseases.

[0027] To achieve the object, the invention indicates a method fordetecting a neurological disease, in particular a progressive,chronically dementia disease, in particular Alzheimer's disease, bydetermination of at least one marker peptide in a biological sample froma patient.

[0028] Various approaches to achieving this are possible and customaryin medical diagnosis:

[0029] On the one hand, it is possible generally to investigate for thepresence of a marker peptide, and the absence or presence of this markerpeptide then makes it possible to diagnose the disease.

[0030] In another customary diagnostic strategy, firstly theconcentrations of the marker peptide which are normally present incontrols and in patients suffering from the disease to be diagnosed aredetermined and, on the basis of these measurements, a limiting value,frequently also called a cut-off point, which separates the groupregarded as healthy from the group regarded as ill is determined. If theconcentration of the particular marker peptide is reduced in people withthe disease, all those whose measurement for the particular markerpeptide is below the cut-off point are diagnosed as having the disease.If the concentration of the particular marker peptide is increased inpeople with the disease, all those whose measurement for the particularmarker peptide is above the cut-off point are diagnosed as having thedisease. The cut-off point determined individually for each markerpeptide thus makes it possible to distinguish healthy people and peoplewith the disease unambiguously.

[0031] In a further diagnostic strategy, an increase in theconcentration or reduction in the concentration, which is specific forthe particular marker peptide, of the marker peptide in the patient'ssample relative to the concentration of the marker peptide in thecontrol sample is determined and a significant marker peptideconcentration change is regarded as positive detection result for thedisease. In this connection, either in principle only an increase in thepeptide concentration of a particular DROPN peptide may occur inpatients with Alzheimer's disease, or in principle only a reduction inthe peptide concentration of this DROPN peptide may occur in patientswith Alzheimer's disease. For a defined DROPN peptide it is not possibleat the same time for an increased DROPN peptide concentration to occurin an individual patient with Alzheimer's disease and for a DROPNpeptide concentration which is reduced relative to the control group tooccur in another patient with Alzheimer's disease.

[0032] Preferred markers according to the invention are indicated in thesequence listing and are named from DROPN-1 to DROPN-31, correspondingto Seq. ID 1 to 31. The sequences of the DROPN peptides are depicted inFIG. 1 and in Table 1. The assignment of the DROPN peptides to theirrespective Seq. ID No. is shown in Table 1.

[0033] The method of the invention comprises a method in which there ismeasurement of specific biomarkers whose concentration is changed inneurodegenerative diseases, especially in Alzheimer's disease, and whichindicate the disease even in a very early stage, e.g. when a minimalcognitive impairment (MCI) is present, or indicate an increased risk ofthe disease at an early date. This is important in order to provide areliable clinical marker for diagnosing these diseases.

[0034] It is possible and preferable for the concentration of DROPNpeptides in the sample, but also the characteristic pattern ofoccurrence of the plurality of particular DROPN peptides, to becorrelated with the severity of the disorder. These novel markerstherefore make it possible to develop and monitor therapies for thetreatment of Alzheimer's disease, because the course and any successfulcure resulting from a therapy or a diminished progression of the diseasecan be established. Effective therapy of Alzheimer's disease is notpossible at present, underlining the urgency for the provision of areliable detection method for Alzheimer's disease, because reliabledetection of the disease is a precondition for the development of atherapy.

[0035] Detection of DROPN peptides additionally makes it possible in theframework of clinical studies to develop novel therapies for thetreatment of Alzheimer's disease with high specificity to select onlythose patients suffering from Alzheimer's disease and not from otherdiseases. This is important for obtaining valid study results. Patientsincorrectly diagnosed as Alzheimer's disease patients have a negativeinfluence on the quality of the results of a study on Alzheimer'sdisease therapy. In addition, detection of DROPN peptides makes itpossible to stratify patients, enabling the specific selection ofsubgroups of Alzheimer's disease patients who are especially suitablefor particular Alzheimer's disease therapeutic strategies or clinicalstudies.

[0036] There are marked changes in the concentrations of DROPN peptidesin Alzheimer's disease patients relative to healthy people. A furtheraspect of the invention is therefore a bringing of the DROPNconcentrations in Alzheimer's disease patients to normal concentrations.This method can be employed for the therapy of Alzheimer's disease orrelated neurological diseases. If the OPN protein or DROPN peptideconcentrations are elevated, the concentrations of these substances canbe reduced by therapeutic administration of, for example, OPN protein-or DROPN peptide-specific antibodies or OPN-specific antisense nucleicacids, ribozymes or triplex nucleic acids for DROPN peptide antagonists,OPN protein antagonists. Substances which suppress the endogenousexpression of OPN protein or the processing of OPN protein to DROPNpeptides can also be administered for the therapy. If the disease iscaused by a deficiency of OPN protein or DROPN peptides, therapeuticdoses of OPN protein, DROPN peptides, DROPN peptide agonists or OPNprotein agonists can be given. Substances which influence the processingof OPN protein to DROPN peptides can also be employed therapeutically.As can be seen in FIG. 1, for example, DROPN-4 and DROPN-10 areseparated from one another by two basic amino acids (lysine andarginine), and such so-called “dibasic sequences” are often the pointsof attack of proteases which are involved in the processing of proteinsto biologically active peptides. Combination of different therapeuticstrategies is, of course, also possible and sensible in somecircumstances.

[0037] The invention therefore also encompasses the use of OPN proteins,DROPN peptides, DROPN peptide agonists and DROPN antagonists, OPNprotein agonists and OPN protein antagonists, anti-OPN proteinantibodies and anti-DROPN peptide antibodies for the direct or indirectmodulation of the concentration of the OPN proteins and DROPN peptidesfor the treatment of neurological diseases, especially Alzheimer'sdisease. Alternative to antibodies, it is also possible to use antibodyfragments, antibody fusion proteins, or other substances which bindselectively to OPN proteins or DROPN peptides. It is also possible asalternative to said proteins and peptides for fusion proteins of saidproteins and peptides to be used. The invention further encompasses alsothe use of antisense nucleic acids, triplex nucleic acids and ribozymeswhich modulate the expression of said proteins and peptides. Theinvention additionally encompasses agonists and antagonists whichmodulate the activity of said proteins.

[0038] A further embodiment of the invention is the pharmaceuticalformulation or chemical modification of the described peptides andnucleic acids to make it possible for them to cross the blood-brainbarrier and/or the blood-CSF barrier more efficiently. They are thusmade particularly suitable for therapeutic use. In order to achievethis, it is possible for example for DROPN peptides, OPN proteins,nucleic acids, agonists or antagonists to be modified so that forexample they become more lipophilic, favoring entry into thesubarachnoid space. This can be achieved by introducing hydrophobicmolecular constituents or else by “packaging” the substances inhydrophobic agents, e.g. liposomes. It is additionally possible forexample for peptide sequences to be attached to these peptides,proteins, nucleic acids, agonists or antagonists, which favor entry intothe subarachnoid space or, conversely, impede emergence from thesubarachnoid space.

[0039] The invention also encompasses the administration of saidtherapeutic agents by various routes such as, for example, asintravenous injection, as substance which can be administered orally, asinhalable gas or aerosol, or administration in the form of directinjection into the subarachnoid space, or into tissue such as muscle,fat, brain etc. It is possible in this way to achieve increasedbioavailability and efficacy of these therapeutic agents. For example,peptides or proteins administered orally can be protected byacid-resistant capsules from proteolytic degradation in the stomach.Very hydrophobic substances can become more hydrophilic and thus bettersuited for, for example, intravenous injections by suitablepharmaceutical processing etc.

[0040] A further embodiment of the invention is the use of DROPNpeptides or of OPN proteins for identifying receptors which selectivelybind these molecules. These receptors can also be modulated byadministration of agonists or antagonists, which is expedient for thetherapy of neurological diseases, especially of Alzheimer's disease.

[0041] OPN Biology

[0042] OPN is synthesized by osteoclasts and osteocytes [2] andincorporated into bone. Osteopontin has been detectedimmunohistologically in the mineralizing zone of developing bones [3].It is additionally present in various biological fluids such as, forexample, urine and milk, and is expressed by activated T cells [4, 5]and by metastasizing tumor cells, elastic fibers of the skin and of theaorta, myocytes, endothelial cells, macrophages and glia cells [6].Detection of OPN in the cerebrospinal fluid has not previously beendescribed and the knowledge about the concentration and the presence ofOPN in the CSF is therefore novel.

[0043] OPN from bovine milk has 28 phosphorylations (27× on serine and1× on threonine), three O-glycosilations and no N-glycosilations [7].Rat OPN isolated from bone has 13 phosphorylations (12× on serine and 1×on threonine) and additionally contains sulfate groups [8]. RecombinantOPN may undergo autophosphorylation on tyrosine. The differences in thenumber of phosphate groups in OPN from bovine milk and OPN from rat boneare presumably based on their different tissues of origin and not fromthe species, because the phosphorylation sites are very highly conservedin all OPN variants sequenced to date [7]. Sorensen et al haveadditionally found that the phosphorylation is almost 100%, i.e. allsites which are phosphorylated are always completely 100% phosphorylated[7]. We have detected OPN in the cerebrospinal fluid for the first time,which has never previously been described in the literature. We havemoreover shown, interestingly, that DROPN-peptides with the samesequence but a different number of phosphorylations occur within thesame sample of cerebrospinal fluid, which was not to be expectedaccording to previous results of OPN in other body fluids and is novel.In addition, only the osteopontin protein has been detected to date inbiological samples, but not osteopontin peptide fragments. During boneremodeling in rats, elevated osteopontin mRNA concentrations occur [9].The connection between age and osteopontin expression is not clearbecause results of different studies describe both an increased and areduced OPN expression in older compared with younger experimentalanimals [2, 9, 10].

[0044] One of the functions of OPN is presumably regulation of crystalgrowth during calcification processes, and the effect of OPN may be bothto enhance and to inhibit calcium crystallization. In atherosclerosisthere is not only calcification of the affected vessel walls but alsoremodeling of the extracellular matrix. Osteopontin can be detectedimmunohistologically preferentially in the calcified regions. [11] andthere is expressed by macrophages and smooth muscle cells. Osteopontinmight possibly serve to regulate vascular calcifications [11]. Thedirection in which osteopontin acts presumably depends on themicroenvironment and the status of osteopontin in relation to itspost-translational modifications, especially its phosphorylation [7].Ek-Rylander et al were able to show, for example, that dephosphorylatedOPN no longer assists osteoclast adhesion [12]. Dephosphorylation of OPNreduces the inhibitory activity of OPN on hydroxyapatite crystalformation, indicating a functional importance of OPN phosphorylation[13]. OPN inhibits crystal growth in the urine and thus prevents thedevelopment of bladder stones. Our results show, however, differing fromthe results described above, that both phosphorylated DROPN peptides andthe corresponding non-phosphorylated DROPN peptides can be used asdementia markers in the same way through their elevated concentration.The markers of the invention are therefore fragments which do notcorrespond to that to be expected for OPN fragments in relation to theirstructural modification. Osteopontin presumably also mediates cell-celland cell-matrix interaction, thus controlling the directed migration ofimmune cells, osteocytes and tumor cells (“homing”) to various sites inthe body. For this purpose, osteopontin interacts with CD44, aubiquitously expressed transmembrane protein [4, 5]. Further ligands ofCD44 besides osteopontin are vitronectin and hyaluronic acid.CD44-osteopontin interaction leads to cellular chemotaxis, whileCD44-hyaluronic acid interaction leads to homotypic cell aggregation. Itwas possible to detect in vitro a chemotactic activity of osteopontin onastrocytes [14]. Osteopontin-deficient mice display disturbances ofwound healing and of the regulation of the immune response [15]. It wasadditionally possible to show that macrophages in the vicinity of humantumors and in necrotic tumor regions, and in ischemic regions of thebrain [14] express large amounts of osteopontin protein and mRNA, andosteopontin therefore presumably has an important function in matrixreorganization during wound healing.

[0045] Osteopontin promotes the adhesion and migration of vascularsmooth muscle cells and endothelial cells. In gliomas, inter aliaosteopontin and its receptor alpha V beta 3-integrin is induced via VEGF(“vasclar endothelial growth factor”), thus possibly inducingangiogenesis. In stroke, which is not a progressive, chronic dementiadisease, it has been possible to show an increase in the OPN mRNA [16].

PREFERABLY EMBODIMENTS OF THE INVENTION

[0046] The dementia detected by the method of the invention ispreferably a progressive, chronic dementia disease such as, for example,Alzheimer's disease. It has been possible to date to detect the changein the concentration of the peptides and peptide fragments of theinvention in various dementia diseases such as, for example, Alzheimer'sdisease or vascular dementia. It can be concluded from this that thepeptides of the invention can also be used for the detection and for thetherapy of Alzheimer's disease and related neurological diseases. Oneembodiment of this method is the determination of dementia diseases atan early date, for example minimal cognitive impairment (MCI).

[0047] The identification is preferably concentrated on particularpeptide fragments of the OPN protein with the GeneBank accession No.X13694, i.e. on peptides which comprise partial sequences of the OPNprotein or else on the OPN protein itself. These peptides (peptidefragments) are referred to as dementia related osteoponton (DROPN)peptides and are referred to hereinafter as DROPN-1 to DROPN-31. Theconnection between OPN protein and DROPN-1 to DROPN-31 is depicted inFIG. 1. The sequences we determined for the peptides are indicated inthe sequence listing. These OPN fragments are produced naturally innature and have not previously been described in the literature. Thesefragments are different from peptides as often described in theliterature, produced by in vitro proteolysis through addition ofproteases such as, for example, trypsin. They therefore represent novel,previously unknown substances. These peptides were initiallyconcentrated and purified from biological samples by reverse phasechromatography and subsequently separated by mass spectrometry fromother accompanying peptides, so that it was subsequently possible tosequence these DROPN peptides.

[0048] The sequences of the peptides in the single-letter amino acidcode are as follows: Mono- OPN isotopic DROPN sequence theoret. No.(X13694) mass (Da) Sequence 1  19-42 2627.2715 VKQADSGSSEEKQLYNKYPDAVAT2  27_(+r1)- * r1-SEEKQLYN-r2  34_(+r2) ≧1009.4716  3 208-243 4032.7594AQDLNAPSDWDSRGKDSYETSQLD DQSAETHSHKQS 4 208-246 4465.0079AQDLNAPSDWDSRGKDSYETSQLD DQSAETHSHKQSRLY 5 211-243 3718.6368LNAPSDWDSRGKDSYETSQLDDQS AETHSHKQS 6 231-245 1737.8030 DDQSAETHSHKQSRL 7231-246 1900.8664 DDQSAETHSHKQSRLY 8 222_(+r3)- ≧956.4087 r3-KDSYETSQ-r4229_(+r4) 9 234_(+r5)- ≧895.4148 r5-SAETHSHK-r6 241_(+r6) 10 249-314 **KANDESNEHSDVIDSQELSKVSRE 7653.6003 FHSHEFHSHEDMLVVDPKSKEEDKHLKFRISHELDSASSEVN 11 249-288 4662.0953 KANDESNEHSDVIDSQELSKVSREFHSHEFHSHEDMLVVD 12 267-283 2093.9304 SKVSREFHSHEFHSHED 13 254_(+r7)-≧899.3985 r7-SNEHSDVI-r8 261_(+r8) 14 271_(+r9)- 1087.4835r9-REFHSHEF-r10 278_(+r10) 15 285-297 1522.7991 LVVDPKSKEEDKH 16 285-2981635.8832 LVVDPKSKEEDKHL 17 285-299 1763.9781 LVVDPKSKEEDKHLK 18 285-3001911.0466 LVVDPKSKEEDKHLKF 19 285-312 3222.6521 LVVDPKSKEEDKHLKFRISHELDSASSE 20 285-314 3435.7634 LVVDPKSKEEDKHLKFRISHELDS ASSEVN 21 286-2991650.8941 VVDPKSKEEDKHLK 22 286-300 1797.9625 VVDPKSKEEDKHLKF 23 286-3123109.5680 VVDPKSKEEDKHLKFRISHELD SASSE 24 289-312 2796.4042PKSKEEDKHLKFRISHELDSASSE 25 290_(+r11 -) ≧1112.5826  r11-KSKEEDKHL-r12297_(+r12) 26 303_(+r13 -) ≧844.3563 r13-SHELDSAS-r14 310_(+r14) 27 19-41 2526.2238 VKQADSGSSEEKQLYNKYPDAVA 28  20-42 2528.2032KQADSGSSEEKQLYNKYPDAVAT 29 211-243 *** LNAPSDWDSRGKDSYETSQLDDQS3718.6368 AETHSHKQS 30 251-285 4149.7995 NDESNEHSDVIDSQELSKVSREFHSHEFHSHEDML 31 251-284 4036.7154 NDESNEHSDVIDSQELSKVSREFH SHEFHSHEDM#scheme explained above, with r3 corresponding maximally to OPN-221-208,r4 maximally to OPN-230-246, r5 maximally to OPN-233-208, r6 maximallyto OPN-242-246, r7 maximally to OPN-253-249, r8 maximally toOPN-262-314, r9 maximally to OPN-270-249, r10 maximally to OPN-279-314,r11 maximally to OPN-289-249, r12 maximally to OPN-298-314, r13maximally to OPN-302-249 and r14 maximally to OPN-311-314. #position ofthe phosphate group in DROPN-10 with one phosphate group is serine atposition 291 of the OPN sequence, the presumed positions of thephosphate groups in DROPN-10 with two phosphate groups are serine 275and serine 291, the presumed positions of the phosphate groups inDROPN-10 with three phosphate groups are serine 270, serine 275 andserine 291. The exact positions of the phosphate groups in DROPN-10 withfour or five phosphate groups is not yet known.

[0049] Suitable Peptides

[0050] The peptides can exist in post-translational or chemicalmodification forms, thus influencing inter alia their masses andtherefore the identification by mass spectrometry and also the eluationbehavior during chromatography, such as, for example, in reverse phasechromatography. In particular, the peptides may be in phosphorylated,glycosilated, sulfated, amidated, oxidized form or with an N-terminalpryoglutamic acid group etc. in the sample to be investigated.

[0051] The peptides are regarded as OPN peptides or DROPN peptides inparticular when a maximum of 30% of their sequence differs from thesequence of the OPN protein. It is permissible in this connection forthere to be point mutations, deletions, insertions and N-terminal and/orC-terminal extensions as long as the difference from the OPN proteinsequence is no more than 30%.

[0052] It is to be assumed that the changes in concentration of themarker peptides (DROPN and OPN peptides) correlate with the severity ofthe disease and the stage of the neurological disease, especially of theprogressive, chronically dementia disease, especially Alzheimer'sdisease. A further development of the invention therefore provides forusing determination of the marker peptides also for determining theseverity and the stage of the disease, in particular as replacement orsupplement to carrying out a mini-mental state examination (MMSE). Afurther development of the invention additionally provides for usingdetermination of the marker peptides for determining preliminary stagesof neurolotical diseases, especially mild cognitive impairment (MCI), orfor prognosis of the course of the disorder.

[0053] The control samples which are possibly used may constitute apooled sample from various controls. The sample to be investigated mayalso be a pooled sample, and where there is a positive result individualinvestigations are subsequently carried out.

[0054] Suitable Biological Samples

[0055] The biological sample may preferably be (human) cerebrospinalfluid (CSF) or a sample such as serum, plasma, urine, stool, tear fluid,sputum, synovial fluid etc. This depends inter alia on the sensitivityof the chosen detection method (mass spectrometry, ELISA etc.). Serum,plasma and urine are particularly of interest because this samplematerial is often obtained without great effort from patients usingstandard investigations. It is also possible where appropriate to usehomogenized tissue samples.

[0056] It is therefore provided in a further embodiment of thisinvention for tissue homogenates to be produced, for example from humantissue samples obtained in biopsies, for preparation of the sample to beinvestigated. These tissues can be comminuted for example with manualhomogenizers, with ultrasound homogenizers or with electrically operatedhomogenizers such as, for example, Ultraturrax, and then be boiled in amanner known to the skilled worker in acidic aqueous solutions with, forexample, 0.1 to 0.2 M acetic acid for 10 minutes. The extracts are thensubjected to the respective detection method, e.g. a mass spectrometricinvestigation. The samples can be prepared, for example whereappropriate diluted or concentrated, and stored in the usual way.

[0057] Use of the DROPN Peptides for Producing Diagnostic Agents

[0058] The invention further comprises the use of at least one of theDROPN peptides of the invention or of a OPN protein for the diagnosis ofneurological diseases, especially chronic dementia diseases, especiallyof Alzheimer's disease, and the use of DROPN peptides for obtainingantibodies or other agents which, because of their DROPNpeptide-specific binding properties, are suitable for developingdiagnostic reagents for detecting these diseases. The invention alsoencompasses the use of DROPN peptides for obtaining phage particleswhich bind these peptides specifically, or which conversely presentDROPN peptides on their surface and thus make it possible to identifybinding partners such as, for example, receptors of OPN proteins orDRBPN peptides.

[0059] Detection Methods for DROPN Peptides

[0060] Various methods can be used for detecting the DROPN peptideswithin the framework of the invention. Methods suitable are those whichmake it possible to detect DROPN peptides specifically in a patient'ssample. Suitable methods are, inter alia, physical methods such as, forexample, mass spectrometry or liquid chromatography, molecular biologymethods such as, for example, reverse transcriptase polymerase chainreaction (RT-PCR) or immunological detection techniques such as, forexample, enzyme linked immunosorbent assays (ELISA).

[0061] Physical Detection Methods

[0062] One embodiment of the invention is the use of physical methodswhich are able to indicate the peptides of the invention qualitativelyor quantitatively. These methods include, inter alia, mass spectrometry,liquid chromatography, thin-layer chromatography, NMR (nuclear magneticresonance) spectroscopy etc. This entails comparison of quantitativemeasured results from a sample to be investigated with the measurementsobtained in a group of patients suffering from neurological diseases, inparticular chronic dementia diseases, preferably Alzheimer's disease,and a control group. It is possible to infer the presence of aneurological diseases, in particular a chronic dementia disease, inparticular Alzheimer's disease, and/or the severity of this disease fromthese results.

[0063] In a preferred embodiment of this invention, the peptides in thesample are separated by chromatography before the identification, inparticular preferably by reverse phase chromatography, with particularpreference for separation of the peptides in the sample byhigh-resolution reverse phase high performance liquid chromatography(RP-HPLC). A further embodiment of this invention is the carrying out ofprecipitation reactions to fractionate the sample using precipitantssuch as, for example, ammonium sulfate, polyethylene glycol,trichloroacetic acid, acetone, ethanol etc. The fractions obtained inthis way are subjected singly to the respective detection method, e.g.the investigation using mass spectrometry. A further embodiment of theinvention is the use of liquid phase extraction. For this purpose, thesample is mixed with a mixture of an organic solvent such as, forexample, polyethylene glycol (PEG) and an aqueous salt solution. Owingto their physical properties, particular constituents of the sample thenaccumulate in the organic phase, and others in the aqueous phase, andcan thus be separated from one another and subsequently analyzedfurther.

[0064] Reverse Phase Chromatography

[0065] A particularly preferred embodiment of this invention encompassesthe use of reverse phase chromatography, in particular a C18 reversephase chromatography column using mobile phases consisting oftrifluoroacetic acid and acetonitrile, for separation of peptides inhuman cerebrospinal fluid. For example the fractions collected in eachcase each comprise {fraction (1/100)} of the mobile phase volume used.The fractions obtained in this way are analyzed with the aid of a massspectrometer, preferably with the aid of a MALDI mass spectrometer(matrix-assisted laser desorption ionization) using a matix solutionconsisting of, for example, of L(−) fucose andalpha-cyano-4-hydroxy-cinnamic acid dissolved in a mixture ofacetonitrile, water, trifluoroacetic acid and acetone, and thus thepresence of particular masses is established and the signal intensityquantified. These masses correspond to the masses of the peptidesDROPN-1 to DROPN-31 of the invention.

[0066] Mass Spectrometry

[0067] In a preferred embodiment of the invention, the peptide(s) can beidentified with the aid of mass spectrometric determination, preferablya MALDI (matrix-assisted laser desorption and ionization) massspectrometry. In this case, the mass spectrometric determination furtherpreferably includes at least one of the following mass signals, in eachcase calculated on the basis of the theoretical monoisotopic mass of thecorresponding peptide. It is possible for slight differences from thetheoretical monoisotopic mass to show owing to the experimental errorand the natural isotope distribution. In addition, in MALDI massdeterminations a proton is added to the peptides owing to the method ofmeasurement, whereby the mass increases by 1 dalton. The followingmasses correspond to the theoretical monoisotopic masses of the peptidesidentified by us, calculated with suitable software, in this case GPMAW4.02. These theoretical monoisotopic masses may occur singly or incombination in a sample: DROPN-1=2627.2715/DROPN-2≧1009.4716/DROPN-3=4032.7594/DROPN-4=4465.0079/DROPN-5=3718.6368/DROPN-6=1737.8030/DROPN-7=1900.8664/DROPN-8≧956.4087/DROPN-9≧895.4148/DROPN-10=7653.6003/DROPN-11=4662.0953/DROPN-12=2093.9304/DROPN-13≧899.3985/DROPN-14≧1087.4835/DROPN-15=1522.7991/DROPN-16=1635.8832/DROPN-17=1763.9781/DROPN-18=1911.0466/DROPN-19=3222.6521/DROPN-20=3435.7634/DROPN-21=1650.8941/DROPN-22=1797.9625/DROPN-23=3109.5680/DROPN-24=2796.4042/DROPN-25≧1112.5826/DROPN-26≧844.3563/DROPN-27=2526.2238/DROPN-28=2528.2031/DROPN-29=3718.6368/DROPN-30=4149.7995and DROPN-31=4036.7154 dalton. The symbol ≧ (is greater than or equalto) is to be understood to mean here that the relevant DROPN peptidescannot have any larger masses but can have only the masses possibleowing to the amino acids which are possibly additionally present at theends of these peptides. Amino acids which may be additionally present atthe ends of these peptides are not just any ones but only those whichmay be present at this sequence position owing to the sequence of theOPN protein.

[0068] Mass Spectrometric Determination of the Sequence of the DROPNPeptides

[0069] For the further practical application of this embodiment, furtherconfirmation of the result of detection is advisable and possible byestablishing the identity of the peptides corresponding to the masses,taking account exclusively of peptide signals which may be derived froman OPN protein. This confirmation takes place by identifying the peptidesignals preferably using methods of mass spectrometry, e.g. MS/MSanalysis [17].

[0070] Novel, specific peptides of OPN proteins (DROPN peptides) wereidentified, and their significance was revealed by the method of theinvention. These DROPN peptides and their derivatives are referred toherein as DROPN-1 to DROPN-31. Their sequences are indicated in thesequence listing. The DROPN peptides DROPN-2, -8, -9, -13, -14, -25 andDROPN-26 may comprise on the N and/or C terminus additional amino acidscorresponding to the corresponding sequence of the relevant OPN protein.The invention also encompasses the DROPN peptides prepared recombinantlyor synthetically, and isolated from biological samples, in unmodified,chemically modified or post-translationally modified form. In thisconnection, two point mutations and other differences are possible aslong as the DROPN peptide has at least 8 amino acids which agree intheir identity and their position within the peptide sequence with anOPN protein.

[0071] Molecular Biology Detection Techniques

[0072] Finally, the invention also encompasses nucleic acids whichcorrespond to DROPN peptides, and especially those which correspond tothe DROPN peptides of the invention, the use thereof for the indirectdetermination and quantification of the relevant OPN proteins andpeptides. This also includes nucleic acids which represent, for example,noncoding sequences such as, for example, 5′- or 3′-untranslated regionsof the mRNA, or nucleic acids which show a sequence agreement with theOPN nucleic acid sequence which is sufficient for specific hybridizationexperiments and which are therefore suitable for the indirect detectionof relevant proteins, especially the DROPN peptides.

[0073] One exemplary embodiment thereof encompasses the obtaining oftissue samples, e.g. of biopsy specimens, from patients and thesubsequent determination of the concentration of an RNA transcriptcorresponding to the gene having the GeneBank accession No. X13694 orcorresponding to homologous OPN variants. This entails comparison ofquantitative measured results (intensities) from a sample to beinvestigated with the measurements obtained in a group of patientssuffering from Alzheimer's disease and a control group. Methods whichcan be used for the quantification are, for example, reversetranscriptase polymerase chain reaction (RT-PCR), quantitative real-timePCR (ABI PRISM® 7700 Sequence Detection System, Applied Biosystems,Foster City, Calif., USA), in situ hybridization or Northern blots in amanner known to the skilled worker. The presence of a chronic dementiadisease, preferably Alzheimer's disease and/or the severity thereof canbe inferred from the results.

[0074] Immunological Detection Methods

[0075] In a further preferred embodiment of the invention, the DROPNpeptides or the OPN proteins can be identified using an immunologicaldetection system, preferably an ELISA (enzyme linked immuno sorbentassay). This immunological detection picks up at least one DROPN peptideor OPN protein. To increase the specificity, it is also possible andpreferred to use the so-called sandwich ELISA in which the detection ofthe DROPN peptides depends on the specificity of two antibodies whichrecognize different epitopes within the same molecule. However, it isalso possible to use other ELISA systems, e.g. direct or competitiveELISA, to detect DROPN peptides or OPN proteins. Other ELISA-likedetection techniques such as, for example, RIA (radio immuno assay), EIA(enzyme immuno assay), ELI-Spot etc. are also suitable as immunologicaldetection systems. DROPN peptides or OPN proteins isolated frombiological samples, recombinantly prepared or chemically synthesized canbe used as standard for the quantification. Identification of the DROPNpeptide(s) is generally possible for example with the aid of an antibodydirected to the DROPN peptide or OPN protein. Further methods suitablefor such detections are, inter alia, Western blotting,immunoprecipitation, dot-blots, plasmon resonance spectrometry (BIACORE®technology, Biacore International AB, Uppsala, Sweden), phage particles,PNAs (peptide nucleic acids), affinity matrices (e.g. ABICAP technology,ABION Gesellschaft fur Biowissenschaften und Technik mbH, Julich,Germany) etc. Substances/molecules suitable as detection agents aregenerally all those permitting the construction of a specific detectionsystem because they specifically bind a DROPN peptide or OPN protein.

[0076] Obtaining of DROPN Peptides and Anti-DROPN Peptide Antibodies

[0077] A further embodiment of the invention is the obtaining of DROPNpeptides using recombinant expression systems, chromatographic methodsand chemical synthesis protocols which are known to the skilled worker.The DROPN peptides obtained in this way can be used inter alia asstandards for quantifying the respective DROPN peptides or as antigenfor producing DROPN peptide antibodies. Methods known to the skilledworker and suitable for isolating and obtaining DROPN peptides includethe recombinant expression of peptides. It is possible to use for theexpression of the DROPN peptides inter alia cell systems such as, forexample, bacteria such as Escherichia coli, yeast cells such asSaccharomyces cerevisiae, insect cells such as, for example, Spodopterafrugiperda (Sf-9) cells, or mammalian cells such as Chinese HamsterOvary (CHO) cells. These cells are obtainable from the American TissueCulture Collection (ATCC). For recombinant expression of DROPN peptides,for example nucleic acid sequences which code for DROPN peptides areinserted in; combination with suitable regulatory nucleic acid sequencessuch as, for example, promoters, antibiotic selection markers etc. intoan expression vector by molecular biology methods. A vector suitable forthis purpose is, for example, the vector pcDNA3.1 from Invitrogen. TheDROPN peptide expression vectors obtained in this way can then beinserted into suitable cells, e.g. by electroporation. The DROPNpeptides produced in this way may be C— or N-terminally fused toheterologous sequences of peptides such as polyhistidine sequences,hemagglutinin epitopes (HA tag), or proteins such as, for example,maltose-binding proteins, glutathione S-transferase (GST), or proteindomains such as the GAL-4 DNA binding domain or the GAL4 activationdomain. The DROPN peptides can be prepared by chemical synthesis forexample in accordance with the Merrifield solid-phase synthesis protocolusing automatic synthesizers which are obtainable from variousmanufacturers.

[0078] A further embodiment of this invention is the isolation of DROPNpeptides from biological samples or cell culture media or cell lysatesfrom recombinant expression systems, e.g. using reverse phasechromatography, affinity chromatography, ion exchange chromatography,gel filtration, isoelectric focusing, or using other methods such aspreparative immunoprecipitation, ammonium sulfate precipitation,extraction with organic solvents etc. A further embodiment of theinvention is the obtaining of monoclonal or polyclonal antibodies usingDROPN peptides. The obtaining of antibodies takes place in theconventional way familiar to the skilled worker. A preferred embodimentof the production and obtaining of DROPN peptide-specific antibodies,and a particularly preferred embodiment is the production of DROPNpeptide-specific antibodies which recognize neo-epitopes, i.e. epitopeswhich are present only on DROPN peptides but not in an OPN protein. Suchanti-DROPN peptide antibodies make the specific immunological detectionof DROPN peptides possible in the presence of OPN protein. Polyclonalantibodies can be produced by immunizations of experimental animals suchas, for example, mice, rats, rabbits or goats. Monoclonal antibodies canbe obtained for example by immunizations of experimental animals suchas, for example mice or rats and subsequent application of hybridomatechniques or else via recombinant experimental approaches such as, forexample, via antibody libraries such as the HuCAL® antibody library ofMorphoSys, Martinsried, Germany, or other recombinant production methodsknown to the skilled worker. Antibodies can also be used in the form ofantibody fragments such as, for example, Fab fragments or Fab2 fragmentsetc.

[0079] Therapy Development and Monitoring Through DROPN PeptideDeterminations

[0080] A further exemplary use is the quantitative or qualitativedetermination of the abovementioned DROPN peptides or OPN proteins forestimating the efficacy of a therapy under development for neurologicaldiseases, in particular chronic dementia diseases, in particularAlzheimer's disease. The invention can also be used to identify suitablepatients for clinical studies for developing therapies for thesediseases, in particular Alzheimer's disease. This entails comparison ofquantitative measured results from a sample to be investigated with themeasurements obtained in a control group and a group of patients. Theefficacy of a therapeutic agent, or the suitability of the patient for aclinical study, can be inferred from these results. The testing ofefficacy and the selection of the correct patients for therapies and forclinical studies is of outstanding importance for successful applicationand development of a therapeutic agent, and no clinically measurableparameter making this reliably possible is yet available for Alzheimer'sdisease [18].

[0081] Examination of the Therapeutic Efficacy of OPN Proteins, DROPNPeptides and of Agents Which Modulate the Expression and theBioavailability of These Substances

[0082] One exemplary embodiment thereof encompasses the cultivation ofcell lines and their treatment with OPN proteins, DROPN peptides or withsubstances which promote the expression of OPN protein or promote theprocessing of OPN protein to DROPN peptides, such as, for example,proteases which recognize dibasic sequence motifs. It is possiblethereby to establish the biological properties of OPN protein and DROPNpeptides in connection with neurological diseases, in particularAlzheimer's disease. Fusion proteins and fusion peptides can also beused for the treatment of the cell lines, e.g. fusion proteins withpeptide sequences which promote transport of the fusion protein into theinterior of the cell. Examples of possible fusion partners are HIV TATsequences or antennapedia sequences etc. It is likewise possible totransfect cell lines with expression vectors which bring about, directlyor indirectly, expression of OPN protein or DROPN peptides by thetransfected cells. These expression vectors may code inter alia forDROPN peptides or OPN proteins. Simultaneous transfections withdifferent DROPN peptides and/or OPN proteins can also be carried out.Alternatively, suitable cell lines can be treated with anti-OPN proteinor anti-DROPN peptide antibodies or with nucleic acids which suppressthe expression of OPN, such as, for example, OPN antisense nucleicacids, OPN triplex nucleic acids or ribozymes directed against OPN mRNA.Cell lines which appear suitable as neurological model systems inconnection with OPN in particular can be used for such investigations.Read-out systems which can be used for these investigations are interalia tests which measure the rate of proliferation of the treated cells,their metabolic activity, the rate of apoptosis of the cells, changes incell morphology, in the expression of cell-intrinsic proteins orreporter genes or which measure the release of cytosolic cellconstituents as markers for cell death. Further test systems which canbe used are suitable strains of experimental animals, e.g. of mice orrats, which are considered as model of neurological diseases, inparticular as model of Alzheimer's disease. These experimental animalscan be used to investigate the efficacy of therapeutic strategies whichaim to modulate the concentration of DROPN peptides or of OPN proteins.It is additionally possible to investigate proteins and peptides suchas, for example, OPN proteins or DROPN peptides in experimental animals,it being possible for these peptides and proteins in some circumstancesto be pharmaceutically processed so that they are better able to crossthe blood-brain barrier and/or the blood-CSF barrier. It is possible touse as pharmaceutical processing method inter alia liposome-packagedproteins and peptides, proteins and peptides covalently fused to ornon-covalently associated with transport peptides such as, for example,an HIV TAT sequence etc. In addition, peptides and proteins can bechemically modified in such a way that they acquire more lipophilicproperties and are therefore able to penetrate more easily into cells.Peptides which are only slightly soluble in aqueous solutions canconversely be chemically modified so that they become more hydrophilicand then can be used for example as intravenously injectable therapeuticagent. Acid-resistant capsules can be used to protect sensitivesubstances, intended for oral administration, in the stomach.

[0083] Read-out parameters in experiments with animal models may be thesurvival time of the animals, their behavior, their short-term memoryand their learning ability. One example of a memory test which issuitable for experimental animals is the Morris water maze test. Furtherparameters which can be used are the determination of body function suchas, for example, blood tests, measurement of brain currents, metabolismtests, the rate of expression of OPN proteins and DROPN peptides andother proteins associated with the disease, and morphological andhistological investigations on tissues such as, for example, the brain.

[0084] The invention is illustrated in detail below by means of examplesreference is also made to the figures in this connection.

[0085]FIG. 1: Alignment of the DROPN peptides with their the OPN protein

[0086]FIG. 2: Reverse phase chromatography for separation and enrichmentof DROPN peptides from cerebrospinal fluid

[0087]FIG. 3: Mass spectrometry measurement (MALDI) on DROPN-10 asexample

[0088]FIG. 4: MALDI as relatively quantifying mass spectroscopic method

[0089]FIG. 5: MS/MS fragment spectrum of the peptide DROPN-10 with onephosphate group as example.

[0090] FIGS. 6A-C: Box-whisker plots for quantitative comparison of theconcentrations of DROPN-5, DROPN-10 and DROPN-20 in patients withAlzheimer's disease compared with control patients.

[0091]FIG. 7: Determination of the concentration of the OPN protein incerebrospinal fluid using a sandwich ELISA.

[0092]FIG. 1 shows an alignment of the OPN peptides of the inventionwith their the OPN protein. The theoretical monoisotopic masses of thepeptides, stated in dalton, were calculated using the GPMAW 4.02software. These are:DROPN-1=2626.2715/DROPN-2≧1009.4716/DROPN-3=4032.7594/DROPN-4=4465.0079/DROPN-5=3718.6368/DROPN-6=1737.8030/DROPN-7=1900.8664/DROPN-8≧956.4087/DROPN-9≧895.4148/DROPN-10=7653.6003/DROPN-11=4662.0953/DROPN-12=2093.9304/DROPN-13≧899.3985/DROPN-14≧1087.4835/DROPN-15=1522.7991/DROPN-16=1635.8832/DROPN-17=1763.9781/DROPN-18=1911.0466/DROPN-19=3222.6521/DROPN-20=3435.7634/DROPN-21=1650.8941/DROPN-22=1797.9625/DROPN-23=3109.5680/DROPN-24=2796.4042/DROPN-25≧1112.5826/DROPN-26≧844.3563/DROPN-27=2526.2238/DROPN-28=2528.2031/DROPN-29=3718.6368/DROPN-30=4149.7995and DROPN-31=4036.7154 dalton. The masses actually identified in themass spectrometer differ from these theoretical, monoisotopic massesbecause of the natural isotope distribution and of a small measurementinaccuracy not exceeding 500 ppm. In addition, the measured mass for allthe peptides is also increased owing to the MALDI measurement methodused by the mass of a proton (=1 dalton). It was additionally possibleto identify and determine experimentally peptide variants having 1 to 5phosphate groups for DROPN-10. The masses experimentally determined forDROPN-10 in this connection are: 7738/7818/7898/7978 and 8058 dalton,with the mass of DROPN-10 being increased sequentially in each case bythe mass of a phosphate group.

[0093]FIG. 2 shows an eluation profile of a with reverse phasechromatography as in Example 2 for the separation and concentration ofthe DROPN peptides from cerebrospinal fluid.

[0094]FIG. 3 shows a spectrum produced by MALDI mass spectrometricmeasurement as in Example 3 of DROPN-10 after reverse phasechromatography of human cerebrospinal fluid as in Example 2. DROPN-10corresponds to the OPN sequence from amino acid 249-314. FIG. 3A showsthe MALDI mass spectrum of DROPN-10 in its non-phosphorylated form. Themass peak of DROPN-10 is marked by an arrow. FIG. 3B shows the MALDImass spectrum of a DROPN-10 variant containing one phosphate group. Themass peak of DROPN-10+1× phosphate is marked by an arrow.

[0095]FIG. 4 shows data generated by MALDI as relatively quantifying MSmethod. A sample was mixed with different amounts of various standardpeptides, and the intensity both of the standard signals and ofrepresentative sample signals was determined. All signal intensities ofthe standards were standardized to their signal intensity at aconcentration of 0.64 μM (=1). Each peptide shows an individual typicalratio of signal strength to concentration, which can be read off in thisdiagram from the gradient of the plot.

[0096]FIG. 5 shows an MS/MS fragment spectrum as in Example 4 of thepeptide DROPN-10 of the invention having one phosphate group.

[0097] Upper trace: raw data of the measurement.

[0098] Lower trace: converted, deconvoluted mass spectrum of DROPN-10having one phosphate group.

[0099] The peak pattern is characteristic of DROPN-10 having onephosphate group. DROPN-10 corresponds to the OPN sequence from aminoacid 249-314.

[0100]FIG. 6 shows box-whisker plots for quantitative comparison of theconcentrations of DROPN-5, DROPN-10 and DROPN-20 in patients withAlzheimer's disease compared with control patients, showing box-whiskerplots for the non-phosphorylated and for mono-, di-, tri- andtetraphosphorylated DROPN-10 peptide. The figures show, in the form ofbox-whisker plots, a comparison of the integrated MALDI massspectrometric signal intensities.

[0101]FIG. 7 shows the results of measurement of the concentrations ofthe OPN protein in cerebrospinal fluid determined using a sandwichELISA, depicted as box plot. The right-hand half of the figure shows theresults of the samples of patients with Alzheimer's disease, the middlepart of the figure shows the results with samples from patients withvascular dementia and the left-hand part of the figures shows theresults of the control group.

EXAMPLE 1 Obtaining Cerebrospinal Fluid for Determining DROPN Peptides

[0102] CSF or cerebrospinal fluid (fluid of the brain and spinal cord)is the fluid which is present in the four ventricles of the brain and inthe subarachnoid space and which is produced in particular in thechoroid plexus of the lateral ventricle. Cerebrospinal fluid is usuallytaken by lumbar puncture and less often by suboccipital puncture orventricular puncture. In lumbar puncture (spinal puncture), to takecerebro-spinal fluid, the puncture involves penetration of the spinalsubarachnoid space between the 3rd and 4th or the 4th and 5th lumbarspinous process with a long hollow needle, and thus CSF being obtained.The sample is then centrifuged at 2000×g for 10 minutes, and thesupernatant is stored at −80° C.

EXAMPLE 2 Separation of Peptides in Cerebrospinal Fluid (CSF) for MassSpectrometric Measurement of DROPN Peptides

[0103] For the detection of OPN peptides in CSF by mass spectrometry, itis necessary in this example to separate the peptide constituents. Thissample pretreatment serves to concentrate the peptides of the inventionand to remove components which may interfere with the measurement. Theseparation method carried out is a reverse phase chromatography. VariousRP chromatography resins and eluants are equally suitable for this. Theseparation of OPN peptides using a C18 reverse phase chromatographycolumn with the size of 4 mm×250 mm supplied by Vydac is [lacuna] by wayof example below. Mobile phases of the following composition were used:mobile phase A: 0.06% (v/v) trifluoroacetic acid, mobile phase B: 0.05%(v/v) trifluoroacetic acid, 80% (v/v) acetonitrile. Chromatography tookplace at 33° C. using an HP ChemStation 1100 supplied by AgilentTechnologies with a micro flow cell supplied by Agilent Technologies.Human cerebrospinal fluid was used as sample. 440 μl of CSF were dilutedwith water to 1650 μl, the pH was adjusted to 2-3, the sample wascentrifuged at 18 000×g for 10 minutes and finally 1500 μl of the sampleprepared in this way were loaded onto the chromatography column. Thechromatography conditions were as follows: 5% mobile phase B at time 0min, from time 1 to 45 min continuous increase in the mobile phase Bconcentration to 50%, from time 45 to 49 min continuous increase in themobile phase B concentration to 100% and subsequently up to time 53 minconstant 100% buffer B. Collection of 96 fractions each of 0.5 ml starts10 minutes after the start of the chromatography. The chromatogram of acerebrospinal fluid sample prepared under the experimental conditionsdescribed herein is depicted in FIG. 2.

EXAMPLE 3 Measurement of Masses of Peptides by Means of MALDI MassSpectrometry

[0104] For mass analysis, typical positive ion spectra of peptides areproduced in a MALDI-TOF mass spectrometer (matrix-assisted laserdesorption ionization). Suitable MALDI-TOF mass spectrometers aremanufactured by PerSeptive Biosystems Framingham (Voyager-DE, Voyager-DEPRO or Voyager-DE STR) or by Bruker Daltonik Bremen (BIFLEX). Thesamples are prepared by mixing them with a matrix substance whichtypically consists of an organic acid. Typical matrix substancessuitable for peptides are 3,5-dimethoxy-4-hydroxycinnamic acid,α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. Alyophilized equivalent obtained by reverse phase chromatography andcorresponding to 500 μl of human cerebrospinal fluid is used to measurethe DROPN peptides of the invention. The chromatographed sample isdissolved in 15 μl of a matrix solution. This matrix solution contains,for example, 10 g/l α-cyano-4-hydroxycinnamic acid and 10 g/l L(−)fucosedissolved in a solvent mixture consisting of acetonitrile, water,trifluoroacetic acid and acetone in the ratio 49:49:1:1 by volume. 0.3μl of this solution is transferred to a MALDI carrier plate, and thedried sample is analyzed in a Voyager-DE STR MALDI mass spectrometerfrom PerSeptive Biosystems. The measurement takes place in linear modewith delayed extraction™. An example of a measurement of one of theDROPN peptides of the invention is shown in FIG. 3.

[0105] The MALDI-TOF mass spectrometry can be employed to quantifypeptides such as, for example, the DROPN peptides of the invention ifthese peptides are present in a concentration which is within thedynamic measurement range of the mass spectrometer, thus avoidingdetector saturation. This is the-case for the measurement of the DROPNpeptides of the invention in cerebrospinal fluid at a CSF equivalentconcentration of 33.3 μl per μl of matrix solution. There is a specificratio between measured signal and concentration for each peptide, whichmeans that the MALDI mass spectrometry can preferably be used for therelative quantification of peptides. This situation is depicted in FIG.4. If various amounts of different standard peptides are added to asample, it is possible to measure the intensity both of these standardsignals and of the sample signals. FIG. 4 shows by way of example aMALDI measurement as relatively quantifying MS method. All signalintensities of the standards were standardized to their signal intensityat a concentration of 0.64 μM (=1). Each peptide shows an individual,typical ratio of signal strength to concentration, which can be read offfrom the gradient of the plot.

EXAMPLE 4 Mass Spectrometric Identification of the DROPN Peptides

[0106] For quantification of the DROPN peptides of the invention it isnecessary to ensure that the mass signals to be analyzed of peptides inthe fractions obtained by reverse phase chromatography of cerebrospinalfluid, as in Example 2, in fact relate to the DROPN peptides of theinvention.

[0107] The peptides of the invention are identified in these fractionsfor example using nanoSpray-MS/MS [17]. This entails a DROPN peptide ionin the mass spectrometer being selected in the mass spectrometer on thebasis of its specific m/z (mass/charge) value in a manner known to theskilled worker. This selected ion is then fragmented by supplyingcollisional energy with an impinging gas, e.g. helium or nitrogen, andthe resulting DROPN peptide fragments are detected in the massspectrometer in an integrated analysis unit, and corresponding m/zvalues are determined (principle of tandem mass spectrometry) [19]. Thefragmentation behavior of peptides makes unambiguous identification ofthe DROPN peptides of the invention possible when the accuracy of massis, for example, 50 ppm by the use of computer-assisted search methods[20] in sequence databases into which the sequence-of an OPN protein hasbeen entered. In this specific case, the mass spectrometric analysistook place with a quadrupole TOF Instrument, QStar-Pulsar model fromApplied Biosystems-Sciex, USA. Examples of MS/MS fragment spectra areshown in FIG. 5.

Example 5 Mass Spectrometric Quantification of DROPN Peptides to CompareTheir Relative Concentration in Control Samples Compared With Patients'Samples

[0108] A sample preparation as in Example 1 and 2 followed by a MALDImeasurement of the DROPN peptides of the invention as in Example 3 werecarried out on 222 clinical samples, i.e. 82 control samples and 130samples from patients suffering from Alzheimer's disease. Examples ofMALDI signal intensities are depicted in the form of box-whisker plotsin FIGS. 6A to 6C. The box-whisker plots depicted in FIG. 6 are based onmeasurements carried out in each case on 29 to 45 samples fromAlzheimer's disease patients, and 13 to 44 control samples perexperiment. A total of 4 experiments was carried out. The box-whiskerplots depicted make it possible to compare the integrated MALDI massspectrometric signal intensities of various DROPN peptides in controlswith the MALDI signal intensities in samples from Alzheimer's diseasepatients. In these, the box, i.e. the columns in the diagrams in FIGS.6A to 6C, in each case includes the range of MALDI signal intensities inwhich 50% of the respective MALDI signal intensities are to be found,and the lines starting from the box and pointing upward and downward(whiskers) indicate the range in which in each case the 25% ofmeasurements which show the highest signal intensities (upper quartile)are to be found, and in which the 25% of measurements. which show thelowest signal intensities (lower quartile) are to be found. The fullline in the columns indicates the median and the broken line in thecolumns indicates the mean.

EXAMPLE 6 Quantification of the OPN Protein With an Enzyme-LinkedImmunosorbent Assay (ELISA) in Human Cerebrospinal Fluid From Patients,and Control Samples

[0109] 20 cerebrospinal fluid samples from patients suffering fromprogressive, chronic dementia diseases and 12 samples from controlsubjects were diluted 1:50 with incubation buffer (140 mM NaCl, 2.7 mMKCl, 1.2 mM KH₂PO₄, 8 mM Na₂HPO₄ 1% bovine serum albumin, 0.05% Tween20) and 100 μm of the samples diluted in this way were put in duplicatesin ELISA plates coated with anti-human OPN antibody 017 (rabbitimmunoglobulin G), and incubated at 37° C. for 1 h. After washing 7times with 200 μl of washing buffer (0.05% Tween 20 in phosphate buffer)each time, 100 μl per well of the secondary antibody which is covalentlycoupled to the enzyme horseradish peroxidase (clone 10A16, monoclonalmouse immunoglobulin G antibody) were incubated in a concentration of 1μg/ml in incubation buffer at 4° C. for 5.5 h, subsequently again washed9 times with washing buffer, and a solution of 0.2 mg/mltetra-methylbenzidine (TMB, Sigma) in substrate buffer (50 mM Na₂HPO₄,20 mM citric acid, pH 5.0) was added as substrate and incubated at roomtemperature with exclusion of light for 30 min. The enzymatic reactionwas stopped by adding 100 μl of stop solution (0.5 M H₂SO₄) per well,and subsequently the absorption was measured at 450 nm in a SUNRISEmodel spectrophotometer from TECAN. A standard series of knownconcentrations prepared with recombinant OPN was determined in the ELISAin parallel and used for the quantification. All the reagents used forthe ELISA were purchased from IBL Hamburg. The OPN concentrations in thecerebrospinal fluid samples, calculated on the basis of the knownconcentrations of the standards, are depicted in the form of box plotsin FIG. 7. Each box includes 50% of the data points with the statisticalmedian as middle line. The upper and lower line of the box indicate thelimits for ±25% of the data population. The line above the upper box isreferred to as upper quartile UQ, and the lower line of the lower box isreferred to as lower quartile LQ. The interquartile distance (IQD)indicates the distance of lower and upper quartile. The lines connectedto the top and bottom of the box indicate the distance to the minimumand maximum respectively. Data points identified as outliers areexcluded from this. This is the case when the value of a data pointW>UQ+1.5*IQD or W<LQ−1.5*IQD.

[0110] The headings in this document are intended merely to providestructure to the text. They are not intended to limit or restrict thematters described. All the examples are intended to characterize theconcept of the invention in more detail but are not intended to restrictthe equivalence range of the invention.

[0111] References

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1. A method for detecting a progressive, chronic dementia disease or apredisposition to such a disease by identifying at least one markerpeptide in a biological sample from an individual, where the markerpeptide is a peptide which is derived from the sequence having the GeneBank accession No. X13694 or a sequence homologous thereto.
 2. Themethod as claimed in claim 1, characterized by determining the relativeconcentration of at least one peptide, compared with the concentrationof the same peptide in a control sample, where a) the concentrationchange, which is specific for the particular marker peptide, in thesample is found relative to a control sample, and b) a significantmarker peptide concentration change in the manner mentioned under b) isregarded as positive detection result for the chronic dementia disease.3. The method as claimed in claim 1 or 2, characterized in that thepeptide a) is a DROPN peptide, b) is a peptide corresponding toaccession No. X13694, or c) is a derivative of a naturally occurringeverything of the peptides mentioned under a) or b), or d) is a DROPNmutant, where the DROPN mutant preferably differs in a maximum of 2,amino acids from the corresponding unmutated DROPN sequence, or e) is amutant of one of the peptides mentioned under b) or c), where the aminoacid sequence differs by a maximum of 30% from the amino acid sequencementioned under b) or c), or f) is a chemically modified, orpost-translationally modified peptide corresponding to a) to e).
 4. Themethod as claimed in any of claims 1 to 3, characterized in that it iscarried out in combination with other diagnostic methods to increase thesensitivity and/or specificity thereof.
 5. The method as claimed in anyof claims 1 to 4, characterized in that the progressive, chronicdementia disease is Alzheimer's disease or a related neurologicaldisease, in particular Lewy body dementia or vascular dementia.
 6. Themethod as claimed in any of claims 1 to 5, characterized in that atleast one identified DROPN peptide is selected, where the peptide is inunmodified form, in chemically modified form or has post-translationalmodifications, preferably as phosphorylated peptide, or with anN-terminal pryoglutamic acid group.
 7. The method as claimed in any ofclaims 1 to 6, characterized in that for a positive detection of thedisease the peptide concentration is increased or reduced for each ofthe peptides in a specific direction relative to the concentration ofthe respective peptide in a control sample.
 8. The method as claimed inany of claims 1 to 7, characterized in that it is used to determine theseverity of the disease, in particular as substitute or supplementcarrying out a mini-mental state examination (MMSE), or to diagnosepreliminary stages of neurological diseases, in particular mildcognitive impairment (MCI), or for prognosis of the course of thedisease.
 9. The method as claimed in any of claims 1 to 8, characterizedin that the biological sample is cerebrospinal fluid, serum, plasma,urine, synovial fluid, sputum, stool, tear fluid or a tissue homogenate.10. The method as claimed in any of claims 1 to 9, characterized in thatthe identification of the peptide(s) is carried out with the aid of amass spectrometric determination, preferably a MALDI (matrix-assistedlaser desorption and ionization) mass spectrometry.
 11. The method asclaimed in claim 10, characterized in that the identification comprisesthe mass spectrometric determination of at least one of the theoretical,monoisotopic mass peaks of2627.2715/≧10.09.4716/4032.7594/4465.0079/3718.6368/1737.8030/1900.8664/≧956.4087/≧895.4148/7653.6003/4662.0953/2093.9304/≧899.3985/≧1087.4835/1522.7991/1635.8832/1763.9781/1911.0466/3222.6521/3435.7634/1650.8941/1797.9625/3109.5680/2796.4042/≧1112.5826/≧844.3563/2526.2238/2528.2031or of 3718.6368 dalton and/or one of the experimentally determinedmasses of 7738/7818/7898/7978 and 8058 dalton.
 12. The method as claimedin any of claims 1 to 9, characterized in that the peptides areidentified with the aid of an immunological, molecular biological,physical or chemical test.
 13. The method as claimed in claim 12,characterized in that the immunological test is an ELISA (enzyme linkedimmuno sorbent assay), a radioimmunoassay or a Western blot.
 14. Themethod as claimed in claim 12, characterized in that the identificationof the peptide(s) is carried out with the aid of an antibody directed toa peptide used according to the invention, of an antibody fragment, of aphage particle, or of PNAs or of an affinity matrix.
 15. The method asclaimed in any of claims 1 to 14, characterized in that the sample isfractionated chromatographically before the identification, preferablyusing reverse phase chromatography, further preferably using highresolution reverse phase chromatography.
 16. The method as claimed inany of claims 1 to 14, characterized in that the sample is fractionatedbefore the identification by precipitation reactions or liquid phaseseparations.
 17. A peptide that a) is a DROP N peptide, b) is a DROPNderivative of a naturally occurring OPN protein, in particular aderivative of X13694, or c) is a DROPN derivative of an OPN allele, ord) is a DROPN mutant, where the DROPN mutant preferably differs in amaximum of 2, amino acids from the corresponding unmutated DROPNsequence, or e) is a chemically, or post-translationally modifiedpeptide corresponding to a) to d).
 18. The use of at least one of theDROPN peptides as claimed in claim 17 for obtaining antibodies and/orfor developing diagnostic reagents for the detection of neurologicaldiseases, in particular of chronic dementia diseases, in particular ofAlzheimer's disease.
 19. Antibodies which bind the DROPN peptides asclaimed in claim
 17. 20. The use of antibodies against osteopontin or ofantibodies as claimed in claim 19 for the diagnosis of neurologicaldiseases, in particular of chronic dementia diseases, in particular ofAlzheimer's disease.
 21. The use of nucleic acids which correspond toDROPN peptides or to OPN proteins for the indirect determination and/orquantification of the relevant proteins and peptides.
 22. The use of amethod corresponding to claim 21, in which the detection of the OPNnucleic acids takes place by using Northern blots, reverse transcriptasePCR or quantitative PCR.
 23. The use of a method as claimed in claims 1to 16, 18 or 20 to 22, for determining the efficacy of a therapy for aneurological disease, in particular for a progressive, chronic dementiadisease, in particular for Alzheimer's disease.
 24. The use of a methodas claimed in claim 1 to 16 or as claimed in claim 20 to 22 forstratifying patients who are suitable for therapies or clinical studiesof neurological diseases, in particular chronic dementia diseases, inparticular Alzheimer's disease.
 25. Nucleic acids which correspond toDROPN peptides.
 26. Nucleic acids which are suitable as OPN-specificantisense nucleic acids or as OPN-specific ribozymes, or as OPN-specifictriplex nucleic acids.
 27. Agonists or antagonists of the OPN peptidesused in claim 1 to
 16. 28. Peptides according to the peptides used inclaim 1 to 16, or substances as claimed in claim 25 to 27, where thesepeptides, nucleic acids, agonists and antagonists are pharmaceuticallyprocessed or chemically or biologically modified in such a way that theyare able to cross the blood-brain barrier and/or the blood-CSF barrier.29. Peptides according to the peptides used in claim 1 to 16, orsubstances as claimed in claim 25 to 27, where these substances arepharmaceutically processed or chemically or biologically modified insuch a way that they are optimized for specific administration routes,in particular for administration into the bloodstream, thegastrointestinal tract, the urogenital tract, the lymphatic system, intothe subarachnoid space, for inhalation or for direct injection intotissue such as, for example, muscle tissue, adipose tissue, brain etc.30. The use of at least one of the peptides used in claim 1 to 16 or ofthe nucleic acids, agonists or antagonists as claimed in claim 25 to 27as medicament or medicament active ingredient.
 31. The use of at leastone of the peptides used in claim 1 to 16 or of the nucleic acids,antagonists or agonists as claimed in claim 25 to 27 for the productionof a medicament for the prophylaxis or treatment of neurologicaldiseases, in particular of chronic dementia diseases, in particular ofAlzheimer's disease.
 32. The use of at least one substance whichmodulates the expression of OPN proteins, for the production of amedicament for the prophylaxis or treatment of neurological diseases, inparticular of chronic dementia diseases, in particular of Alzheimer'sdisease.
 33. The use of a substance which binds to at least one of thepeptides used in claim 1 to 16, in particular of antibodies, antibodyfragments, PNAs or affinity matrices for the production of a medicamentfor the prophylaxis or treatment of neurological diseases, in particularof chronic dementia diseases, in particular of Alzheimer's disease. 34.The use of at least one of the peptides used in claim 1 to 16 or of thenucleic acids, antagonists or agonists as claimed in claim 25 to 27 forthe therapy of neurological diseases, in particular of chronic dementiadiseases, in particular of Alzheimer's disease.
 35. A method for thetherapeutic modulation of the concentration of at least one of thepeptides used in claim 1 to 16 or of nucleic acids as claimed in claim25 in a patient with a neurological disease, in particular chronicdementia diseases, in particular Alzheimer's disease.
 36. A methodcorresponding to claim 35, in which a reduction in the concentrations ofOPN peptides, DROPN-peptides or OPN nucleic acids is desired.
 37. Amethod corresponding to claim 35, in which an increase in theconcentrations of OPN proteins, DROPN peptides or OPN nucleic acids isdesired.
 38. A method corresponding to claim 36, in which a) antibodiesdirected against OPN proteins or DROPN peptides are administered, b)antisense nucleic acids, triplex nucleic acids or ribozymes areadministered, in order to reduce the expression of OPN proteins or DROPNpeptides, or c) substances which inhibit the processing of OPN proteinsare administered, or d) antagonists of the OPN peptides used in claim 1to 16 are administered to a patient.
 39. A method corresponding to claim37, in which a) OPN proteins or DROPN peptides are administered, or b)nucleic acids which code for OPN proteins or DROPN peptides areadministered, or c) substances which promote the processing of OPNproteins are administered, or d) agonists of the OPN peptides used inclaim 1 to 16 are administered to a patient.
 40. A screenig method foridentifying substances able to reduce or enhance the expression of atleast one of the peptides used in claim 1 to
 16. 41. A screening methodfor identifying receptors, or inhibitors which bind at least one of thepeptides used in claim 1 to
 16. 42. A screening method for identifyingagonists or antagonists, of at least one of the peptides used in claim 1to 16.